Gastric aspiration is a major risk factor for the development of acute lung injury (ALI) and ARDS. This proposal examines cellular mechanisms that increase the risk of aspiration-associated pathology. Although many host changes are poorly understood, our prior work has demonstrated a role forTNFalpha, IL-1U, IL- 6, IL-10, CXC chemokines, MCP-1, and toxic inflammatory products in the pathogenesis of aspiration- induced ALI. We have also demonstrated that low pH secretions in the gastric material synergistically increase the acute severity and sustainability of ALI following a subsequent insult (i.e., small food particles) Aim 1 will utilize drug-induced alveolar macrophage (aM0) depletion and reconstitution, transgenic (gene deletion and over-expression) mice, and flow cytometric strategies to examine in detail the role resident and recruited aM0, and alveolar epithelial cells (AEC) play in the pathogenesis of the severe ALI induced by combined acid and small particles (CASP). We hypothesize that the interaction of the components of CASP on aM0 and AEC responses leads to a synergistic, sustained ALI with accelerated aM0 apoptosis/necrosis and recruitment of a new M0 population with altered cytokine expression profiles. The role of the Fas/FasL system and apoptosis of recruited leukocytes in the pathogenesis of CASP ALI will also be assessed. Aim 2 will examine the roles of specific mediators of the inflammatory transition, including studies using recombinant IL-6, IL-10, MCP-1, TNFalpha, or over expression of these cytokines with transgenic mice or defective adenovirus vectors, or by employing anti-cytokine antibodies or gene deletion mice. We predict that TNFalpha-induced IL-6, IL-10, and/or MCP-1 cytokines play an important role in the transition from an acute to a less intense inflammatory response following gastric aspiration. In the final aim, we will employ a proteomic approach utilizing a novel multiplex microarray ELISA technology to identify local and systemic biomarkers and aM0 phenotypes to compare responses in patients and mice in order to provide mechanistic insight into the etiology and pathogenesis of aspiration-induced ALI and provide a rationale on which to test therapeutic strategies. Additionally, this will provide pilot data for developing diagnostic and prognostic biomarkers to differentiate aspiration events from bacterial pneumonia.